A global public health goal is the control and eventual eradication of human malaria, which is caused primarily by one of four species of the Plasmodium parasite: P. falciparum, P. vivax, P. malariae, and P. ovale. It is estimated that over 500 million people in tropical regions are exposed to malaria annually, and 1.5 to 2 million people die from this disease. Efforts to control malaria have historically focused on control of the mosquito vector and the development of anti-malarial drugs. These efforts have met with only limited success. New prophylactic and therapeutic drugs are of limited effectiveness because drug-resistant strains can appear rapidly in endemic areas. Control of the mosquito vector depends largely upon implementation of insecticide-based control programs which, due to cost and other factors, are difficult to maintain in developing nations. Vector resistance to modern insecticides has compounded the problem, and resulted once again in the resurgence of malaria.
Mammalian hosts can be infected by the sporozoite form of the malaria parasite, which is injected by the female Anopheles mosquito during feeding. Sporozoites injected into the bloodstream are carried rapidly to the liver where they invade hepatocytes, the beginning of liver-stage infection. Once in hepatocytes, sporozoites develop into merozoite forms, which are released from hepatocytes and invade erythrocytes. Within the erythrocyte, the parasite asexually reproduces from rings to schizonts. This stage of the parasite's life cycle is known as the blood-stage. The mature schizont contains merozoites which, upon rupture of the erythrocyte, can invade other erythrocytes, causing clinical manifestations of the disease. Some merozoites differentiate into sexual forms, called gametocytes, which are taken up by mosquitoes during a blood meal. After fertilization of gametocytes in the mosquito midgut, developing ookinetes can penetrate the gut wall and encyst. Rupture of such oocysts allows release of sporozoites that migrate to the salivary glands to be injected when the female mosquito takes another blood meal, thus completing the infectious cycle. This stage, which occurs within the mosquito, is called the extrinsic cycle or mosquito stage.
Experiments conducted in the 1960s demonstrated that vaccination with X-irradiated sporozoites of Plasmodium berghei (P. berghei) protected mice against sporozoite challenge, which was lethal in unvaccinated animals. This observation was later extended to clinical studies in humans where immunization with X-irradiated sporozoites of P. falciparum or P. vivax protected human volunteers against sporozoite challenge delivered through the bites of infected mosquitoes. This protection was thought to be mediated by antibodies. Serum from immunized animals, including humans, formed a precipitate around the surface of live, mature sporozoites. This reaction has been termed the circumsporozoite precipitin (CSP) reaction. These same sera blocked the ability of sporozoites to invade human hepatoma cells in culture (ISI assay). In other studies, a single antigenic determinant localized on the surface of P. berghei sporozoites, termed the circumsporozoite protein, was identified. It was shown that a monoclonal antibody reacting with the circumsporozoite (CS) protein of P. berghei could passively transfer immunity to recipient animals. These animals were protected from sporozoite challenge in a dose-dependent fashion. Evidence also existed that cell-mediated immunity was important.
The first CS protein gene to be cloned was derived from the H strain of P. knowlesi, a simian parasite. The genes encoding the CS proteins of the human malaria parasites P. falciparum, P. vivax, the simian parasite P. cynomolgi, and the rodent parasite P. berghei were also cloned and sequenced. A characteristic feature of the CS genes of each of the parasites is a central region which encodes over one-third of the protein, containing a series of repeated peptide sequences. The primary amino acid sequence, the length of the repeated sequence, and the number of repeats vary with each species of parasite. The repeat region epitopes are characteristic of each species. The gene encoding the CS protein of P. falciparum specifies a central repeat region of a tetrapeptide (asn-ala-asn-pro) repeated 37 times, interrupted in four locations by the nonidentical tetrapeptide (asn-val-asp-pro). The central repeat region of P. vivax CS protein contains 19 nonapeptides; the central sequence of P. knowlesi contains 12 dodecapeptides, and the repeat region of P. berghei contains 12 octapeptides. Comparison of sequences from P. knowlesi (H strain) and P. falciparum and P. vivax reveals no sequence homology except for two short amino acid sequences flanking the repeat region, termed Region I and Region II.
Efforts to develop an effective anti-sporozoite vaccine for P. falciparum have used peptides derived from the circumsporozoite (CS) repeat region and the two flanking Region I and Region II sequences. These experiments showed that antibody to the repeat region but not to the conserved sequences recognized authentic CS protein, produced CSP activity, and blocked sporozoite invasion (ISI) in vitro. A recombinant DNA subunit vaccine composed of 32 P. falciparum tetrapeptide repeats fused to 32 amino acids of the tetracycline resistance gene was produced in E. coli. Likewise, a peptide-carrier vaccine composed of three repeats of the peptide asn-ala-asn-pro (NANP) conjugated to tetanus toxoid was developed. In each case, preclinical studies indicated that biologically active (as shown by CSP and ISI) anti-sporozoite antibodies were elicited as a result of immunization. Human safety and immunogenicity studies with both vaccines yielded similar results. Both vaccine preparations were well tolerated at doses ranging from 10 micrograms to 800 micrograms, and both elicited some anti-CS antibodies in all immunized subjects. However, high titers were not achieved. In addition, subsequent booster immunizations with the peptide-carrier vaccine did not result in increased antibody titers. Several individuals from each study were then challenged with live sporozoites in order to test the efficacy of these vaccine preparations. Once again, similar results were achieved with both vaccines; the level of protection (as measured by a delay in the appearance of blood stage parasites) correlated with the anti-CS antibody titers of the challenged individuals, but in each trial, only one individual was protected. Parallel studies to evaluate the feasibility of human subunit vaccine development have been examined in the rodent P. berghei malaria model.
Another study has reported that levels of naturally acquired antibodies to the P. falciparum CS protein, as high as those achieved by a subunit sporozoite vaccine, did not protect against P. falciparum infection during a 98-day interval in a malaria-endemic area.
In different studies, subunit vaccines containing peptides of other P. falciparum antigens have been investigated. In addition, recombinant vaccinia viruses, which express P. falciparum antigens, have been described for use. More current approaches to malaria vaccines also include DNA vaccines, a malaria specific protein developed using transgenic technology as a vaccine, as well as genomic and proteomic approaches.
Perspectives and advances in malaria vaccination have been described (Miller, L. H., et al., 1984, Phil. Trans. R. Soc. Lond. B307:99-115; 1985, VACCINES 87, Channock et al., eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. pp. 81-106, 117-124). More recent advances in malaria vaccines are described in a review paper entitled “Malaria Vaccine Development: Status Report” (S. James and L. Miller, Institute of Allergy and Infectious Diseases, NIH, Washington D.C.: GPO 2000), which can also be obtained on the website of the Division of Microbiology & Infectious Diseases, National Institute of Allergy and Infectious Disease, NIH.
There are many scientific questions that must be addressed in the course of further development efforts on malaria vaccines. These include issues such as how to induce appropriate (protective, long-lasting, nonpathogenic) immune responses, how to structure combination vaccines, how to deal with parasite antigen diversity and antigenic variation, as well as how to deal with human genetic restriction of immune response and/or genetic predilection toward detrimental responses.
There are also a number of hurdles related to research and evaluation of candidate vaccines. These include issues regarding the appropriateness and accessibility of animal models. Other technical hurdles relate to the need to identify assays for ongoing validation of candidate antigens through process development and scale-up production, as well as assays predictive of protection for assessment of immunogenicity and efficacy in clinical trials. In addition, much careful thought must be given to clinical trial design. This is especially true for blood-stage vaccines, where the feasibility of experimental challenge infection is extremely controversial and the optimal measurements of efficacy is reduced morbidity/mortality, as well as for sexual stage vaccines, where the ultimate measurement of efficacy is interruption of malaria transmission.
The development and widespread availability of highly effective attenuated malarial vaccines to provide efficacious immunization against malaria would be highly desirable and beneficial for humans at risk of contracting the disease. No approved malaria vaccine is currently available, and currently available drugs used to treat malaria are only partially effective.